An in vitro nutritive evaluation of Medicago arborea as affected by growth stage and cutting regimen

M R Al-Masri

Department of Agriculture, AEC, PO Box 6091, Damascus, Syria
ascientific@aec.org.sy

Abstract

Nutritive values of the branches of Medicago arborea cut at 25 or 50 cm distance from the tip (cutting length) and at five different growth stages (vegetative, early flowering, fruiting, dormant and vegetative re-growth) were evaluated by determination of the in vitro digestible organic matter (IVDOM), metabolizable energy (ME) and net energy lactation (NEL) and presence of nutritional and anti-nutritional components. The sampling or harvest stages was conducted at mid winter, early spring, late spring, late summer and mid autumn for each of the five growth stages, respectively.

The values of nitrogen forms, ash, IVDOM and ME declined and concentrations of neutral-detergent fiber and acid-detergent fiber increased in the fruiting and dormant stages compared with other growth stages and between cutting length at 25 cm and 50 cm (P < 0.05). Increasing the cutting length from 25 to 50 cm negatively affected all the studied nutritive parameters. In autumn, winter and early spring, branches of M. arborea showed higher contents of crude protein (158 g/kg DM), IVDOM (677 g/kg DM) and ME (9.25 MJ/kg DM) than other seasons. The evaluated samples had low concentrations of hydrolysable and condensed tannins. There were no differences in the values of IVDOM, ME and NEL due to addition of polyethylene glycol to the plant samples incubated with rumen fluid. IVDOM, ME and NEL values were negatively correlated with cell wall constituents but positively correlated with nitrogen forms. Results revealed that, the harvested branches of M. arborea at the vegetative, early flowering and vegetative re-growth stages were better in terms of nutritional value than those harvested at dormant and fruiting stages.

Key words: arid steppe, feed, harvesting, legume, nutritional value, polyethylene glycol

Introduction

Shrubs are important in grazing systems as a feed source for livestock, especially during the dry season when herbaceous species absent and do not meet animal nutrient requirements (Lefroy et al 1992; Douglas et al 1996). Medicago arborea L. is one of the most potentially valuable fodder shrubs in Mediterranean regions because of its high preference by small ruminants (Amato et al 2004). It is a woody leguminous shrub that belongs to the Fabaceae family, and grows on dry calcareous soils.

The  chemical composition of shrubby species intended to be used for forage is closely related to their feeding value. The seasonal variation of forage composition results from physiological changes which occur in plants during their growing seasons. However, species vary in their response to climatic and physiological changes (Dann and Low 1988), and these differences determine their practical value as forage shrubs. The nutritive quality of the forage and its content of anti-nutritional components are influenced by harvest time (Yihalem et al 2005; Al-Masri and Mardini 2008; Nordheim-Viken and Volden 2009; Nordheim-Viken et al 2009; Foster et al 2012) and  cutting regimen (Čop et al 2009; Al-Masri 2009, 2010). The phenolic compounds (particularly tannins) in some shrubs and roughages may bind to protein, thus rendering the protein undegradable by rumen microbes. Polyethylene glycols (PEG) is able to form complexes with tannins (Getachew et al 2000) and has been used to reduce tannin-protein complex formation or to release these complexes (Makkar et al 1995).

Objectives

The objectives of the present study were:

• To evaluate branches of M. arborea forage samples cut at different growth stages (vegetative, early flowering, fruiting, dormant and vegetative re-growth) and at 25 or 50 cm distance from the tip in terms of their contents of nitrogen forms, cell wall constituents and nutritional and anti-nutritional components.

• To measure in vitro digestible organic matter and metabolizable energy of the leaves of  experimental branches as well as their impact on the net energy lactation.

•  To study the influence of polyethylene glycol on these parameters using an in vitro ruminal gas production technique.

Materials and Methods

Experimental design and sampling of materials

The study was conducted using a 5 X 2 factorial experiment arranged in a randomised block design with 4 replicates. Branches of 3-year-old plants of M. arborea, grown at Der Al-Hajer research station about 30 km south east of Damascus in the arid steppe region, which receives a total annual precipitation of 120-130 mm, were hand-cut at five growth stages (vegetative, early flowering, fruiting, dormant and vegetative re-growth) at 25 or 50 cm distance  from the tip (cutting length) with 4 replicates (n = 4) (4 plants each). Sampling or harvest stages was conducted at mid winter, early spring, late spring, late summer and mid autumn for each of the five growth stages previously mentioned, respectively. The plants were irrigated 6 times during the summer season. The sampled branches for each replicate were mixed individually and three representative sub-samples, 2.5 kg each, were randomly chosen, dried at room temperature (20-25 ^oC) for one week, ground to pass through a 1-mm sieve and stored frozen at -20 ^oC in sealed nylon bags for later analyses. In addition, 3 representative sub-samples, 2 kg each, were also taken randomly from each mixed sample and divided by hand into leaves and stalks.

Determination of nutritional and anti-nutritional components

Standard methods as described in AOAC (1990) were used for determination of dry matter (DM), ash, ether extract (EE) and crude protein (CP). Cell-wall constituents (neutral-detergent fiber, NDF; acid-detergent fiber, ADF and lignin) were analyzed (Van Soest et al 1991).

Buffer soluble nitrogen (BS-N) and non-protein nitrogen (BS-NPN) were determined according to Makkar and Becker (1996). 50 mL of phosphate buffer (0.05 M, pH 7.0) in a centrifuge tube was added to 2.5 g sample and the contents were ultra-turraxed at 10 000 rpm for 4 x 3 min with intermittent cooling. The supernatant liquid was separated by centrifuging at 3500 g for 15 min. The buffer-soluble nitrogen was determined on aliquots of the supernatant by the Kjeldahl method. To other 10 mL aliquots an equal volume of 20% trichloroacetic acid was added; the mixture was kept overnight in a refrigerator and centrifuged at 3500 g for 15 min to collect the protein-free supernatant, aliquots of which were analyzed for non-protein nitrogen.

Total phenols (TP), hydrolysable tannins (HT) and condensed tannins (CT) were determined by spectrophotometric methods. Total phenols were quantified by Folin Cio-calteu reagent and hydrolysable tannins as the difference of phenolics before and after tannin removal from the extract using insoluble polyvinylpyrolidone (Makkar et al 1993). Condensed tannins were determined by the butanol-HCL method (Porter et al 1986).

Estimation of digestible organic matter, metabolizable energy and net energy lactation

In vitro digestible organic matter (IVDOM) and metabolizable energy (ME) and net energy lactation (NEL) were estimated according to the methods of Menke et al (1979) using a gas production technique, by incubating samples in 100 mL calibrated glass syringes at 39 ^oC with a rumen fluid mixture for 24 h, and with or without adding polyethylene glycol at a ratio of 2:1 PEG:substrate to study the biological activity of tannins (Makkar et al 1995). The equations used to estimate the IVDOM, ME and NEL values were:

IVDOM (g/kg DM)  =  [16.49 + 0.9042 (mL gas produced) + 0.0492 (protein g/kg DM) + 0.0387 (ash g/kg DM)] 10

ME (MJ/kg DM)  =  2.20  +  0.1357 (mL gas produced) + 0.0057 (protein g/kg DM) + 0.0002859 (lipid g/kg DM)²

NEL (MJ/kg DM) = 0.54 + 0.0959 (mL gas produced) + 0.0038 (protein g/kg DM) + 0.0001733 (lipid g/kg DM)²

The equations for roughages were chosen according to Menke and Steingass (1988). The volume of gas was based on that produced by incubating 200 mg of substrate for 24 h compared with that produced by the standard hay sample (Hohenheim University, Germany) used by Steingass and Menke (1986) to control quality of the rumen fluid.

The rumen fluid was collected before the morning feeding from 3 rumen-fistulated Awassi rams to avoid changes in rumen fluid activity during the experiment. The fastulated rams were principally fed on a roughage diet and received 162 g crude protein and 12.8 MJ ME per day. Rumen fluid samples were taken once every 7 days, 16 h after the last meal. The rumen fluid was homogenised and strained through 100-µm nylon cloth into a warm flask (39 ^oC) filled with CO₂. A total of 30 mL medium, consisting of 10 mL of rumen fluid and 20 mL of bicarbonate-mineral-distilled water mixture (1: 1: 2 by vol.), was pumped with an automatic pipette into the warmed syringes containing the samples (200 mg) and into the blank syringes. The syringes were shaken by hand for a couple of seconds, twice in the first hour and once again after 3, 6, and 8 h of incubation. Gas production from the experimental sample was recorded after 24 h of incubation and calculated by subtracting the volume of gas produced from the blank with or without the addition of PEG.

Statistical analyses

Results were subjected to a factorial analysis of variance (ANOVA) test, using a Statview-IV program (Abacus Concepts, Berkeley, CA, USA). The three main factors were: growth stage (vegetative, flowering, fruiting, dormant and vegetative re-growing), cutting length (25 and 50 cm) and polyethylene glycol treatment (PEG or no PEG). Means were separated using the Fisher’s least significant difference test at the 95% confidence level. Regression coefficients (R) between the studied parameters were calculated.

Results and Discussion

Chemical composition

Cutting length of the branches of M. arborea positively affected the neutral-detergent fiber (NDF), acid-detergent fiber (ADF) and lignin and negatively affected the crude protein (CP), ether extract (EE) and ash levels (Table 1). The values of CP, EE and ash declined and NDF, ADF and lignin increased between cutting length of branches at 25 and 50 cm. There was a negative correlation between the CP values and the NDF concentrations (R = -0.74; P < 0.001), ADF (R = -0.55; P < 0.001) and lignin (R = -0.60; P < 0.001). Chemical composition of the branches of M. arborea were significantly (P < 0.05) affected by the growth stage (Table 1). The values of  CP decreased and concentrations of NDF and ADF increased in the fruiting and dormant stages compared with other growth stages (P < 0.05).

The CP concentrations in all treatments were well above the minimum of CP level of 75 g/kg DM required for rumen microorganisms function (Van Soest 1994). The average crude protein (158 g/kg DM) value of the branches of M. arborea harvested at vegetative, early flowering and vegetative re-growing stages was lower than those reported by Sallam et al (2008) for alfalfa hay (182 g/kg DM) and comparable or higher than those reported by El-Waziry (2007) for trefoil hay (145 g/kg DM) as a commonly used forage for livestock. A minimum of 150 g crude protein per kg DM is required for lactation and growth of cattle (Norton 1982); in the present study the former crude protein (158 g/kg DM) value of M. arborea was comparable to this recommended value. The harvested plants at fruiting and dormant stages had higher concentrations of EE and lignin than other growth stages, respectively. In autumn, winter and early spring, branches of M. arborea showed higher contents of crude protein and lower concentrations of NDF and ADF than other seasons (late spring and late summer). Parissi et al (2005) indicated that crude protein decreased while NDF and ADF and ADL increased in some browse species (Medicago arborea, Arbutus andrachne and Gleditsia triacanthos) as a result of maturation. Narvaez et al (2010) showed that crude protein content of 11 California browse species decreased from spring to fall.

The changes in nitrogen forms and anti-nutritional components of the branches of M. arborea, as affected by cutting length and growth stage are shown in Table 2 and Figures 1 and 2. Increasing cutting length from 25 to 50 cm negatively affected the BS-N, BS-NPN and total phenols values (P < 0.05). The BS-N and BS-NPN concentrations were higher (P < 0.05) in branches cut at 25 cm length compared with 50 cm cutting length, indicating a higher solubility of nitrogen at the former length (25 cm) which included a higher amount of leaves (0.44) than those cut at the 50 cm length (0.31 of leaves). Al-Masri and Mardini (2008) indicated that leaves of Kochia indica and Sesbania aculeate had higher concentrations of BS-N and BS-NPN than stalks. Time of harvesting had a significant (P < 0.05) effect on the values of BS-N, BS-NPN and phenols. The harvested branches of M. arborea at fruiting and dormant stages had lower concentrations of BS-N, BS-NPN and total phenols than other growth stages.

Figure 1.  Changes in buffer soluble nitrogen, buffer soluble non- protein nitrogen and total phenols in harvested branches of Medicago arborea, as affected by length of branch	Figure 2.  Changes in buffer soluble nitrogen, buffer soluble non-protein nitrogen and total phenols in harvested branches of Medicago arborea, as affected by growth stage	Figure 3.  Changes in hydrolyzable and condensed tannins in harvested branches of Medicago arborea, as affected by growth stage

The experimental plant samples had very low concentrations of hydrolysable (HT) and condensed tannins (CT) (Fig. 1). The average amounts of total phenols (TP), HT and CT in the samples were 10.94, 0.16 and 0.37 g/kg DM, respectively. Getachew et al (2002) reported that plant samples containing total phenols and tannin levels (g tannic acid equivalent/kg DM) up to 40 and 20, respectively, were not expected to precipitate protein or cause increases in gas production upon addition of PEG to the in vitro ruminal gas production method and, therefore, are not likely to adversely affect ruminant productivity. Values of total condensed tannins exceeding 50 g/kg DM could inhibit microbial activity and depress dry matter digestibility (Kumar and Vaithiyanathan 1990).

In vitro digestible organic matter (IVDOM),  metabolizable energy (ME) and net energy lactation (NEL)

The effects of growth stage and cutting length, and their interaction, on the IVDOM, ME and NEL levels of M. arborea after incubation with or without polyethylene glycol (PEG) are illustrated in Table 3. The differences in IVDOM, ME and NEL values of the branches of M. arborea affected by growth stage and cutting regimen reflect different contents of nutritive components in the experimental samples. The IVDOM, ME and NEL values were negatively correlated with NDF and ADF (R = -0.85; P < 0.001) and lignin concentrations (R = -0.71; P < 0.001) and positively correlated with CP, BS-N and BS-NPN concentrations (R = 0.82; P < 0.001). Khanum et al (2007) indicated that feedstuffs (wheat straw, grasses and crop residues) having different digestibility coefficients of organic matter showed differences in ME and that the ME values were very low in feedstuffs having high fiber and low protein contents.

Lignocellulosic materials, particularly lignin, act as a physical barrier to microbial enzymes. It is generally agreed that the lignin concentration of forages is negatively related to the extent of digestion (Jung et al 1997). The lignin suppressing effect probably results from a reduction in attachment of ruminal microbes to feed particles and inhibition of microbial enzyme activity (McSweeny et al 2001). 

Results indicated that increasing cutting length from 25 to 50 cm negatively affected the IVDOM, ME and NEL values (P < 0.05). Zarkawi et al (2005) reported that the IVDOM and ME of leaves of Sesbania aculeate were higher than that of stalks. However, in a study with 11 California chaparral browse species, Narvaez et al (2010) showed that leaves of all species had higher concentrations of crude protein, in vitro organic matter digestibility and ME and lower concentrations of NDF and ADF compared to stalks. Leaves have a low content of lignocellulosic materials and are usually more digestible (i.e. more fermentable by rumen microorganisms) than stalks. Therefore, the cutting branches of M. arborea at 25 cm length, which included a greater amount of leaves (0.44), had higher values of IVDOM, ME and NEL than those cutting at the 50 cm length (0.31 of leaves). The average proportion of leaf (dry matter basis) in the harvested branches at vegetative, early flowering, fruiting, dormant and vegetative re-growth stages amounted to 0.44, 0.54, 0.33, 0.16 and 0.39, respectively.     

The harvested branches of M. arborea at vegetative, early flowering and vegetative re-growth stages with cutting performed at 25 cm distance from the tip gave higher values of IVDOM and ME than the other growth stages (fruiting and dormant). In autumn, winter and early spring, branches of M. arborea showed higher contents of IVDOM (677 g/kg DM) and ME (9.25 MJ/kg DM) than other seasons (late spring and late summer). The former IVDOM value of M. arborea was higher than those (447 g/kg DM) reported by Sallam et al (2008) for alfalfa hay. Long et al (1999) showed that ME of 22 native forages of China harvested in August, September and October decreased with maturity. The total indigestible neutral-detergent fiber content increased with maturity stage of Phleum Partense (Nordheim-Viken and Volden 2009). Ammar et al (2004) indicated that crude protein content, digestibility coefficient and parameters of gas production kenetics of some leguminous shrub species (Cytisus scoparius, Genista florida and Genista scorpius) tended to decrease from spring to autumn, whereas cell wall contents followed the opposite trend.  

The addition of polyethylene glycol in the fermentation process did not increase the IVDOM, ME and NEL values of the branches of M. arborea (Table 2). This might be related to the low concentrations of condensed tannins in the experimental samples. Addition of PEG can be useful if the tannin content of the feed is adequately high to the extent that it depresses microbial activity and digestibility of feeds drastically (Getachew et al 2000).

Conclusions

Based on the results of this research it is concluded that:

• Crude protein, ash, BS-N, BS-NPN, IVDOM and ME contents of the branches of M. arborea tended to decrease at the fruiting and dormant growth stages, whereas NDF and ADF contents followed an opposite trend. Increasing the cutting length from 25 to 50 cm negatively affected all the evaluated nutritive parameters.
  
  
• The experimental plant samples had low concentrations of hydrolysable and condensed tannins. Therefore, addition of PEG to the experimental samples did not increase the values of IVDOM, ME and NEL. 
  
  
• The harvested branches of M. arborea at the vegetative, early flowering and vegetative re-growth stages with cutting performed at 25 cm distance from the tip were judged to be nutritionally better and more suitable as dietary supplements for livestock in Mediterranean regions than those harvested at dormant and fruiting stages.

Acknowledgements

The author thanks the Director General and Head of Agriculture Department, A.E.C. of Syria, for their encouragement and financial support.

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Received 19 February 2013; Accepted 19 February 2013; Published 1 May 2013

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